Development of New Molecular Tools for Antibody Detection and Purification

Abstract

Primary antibody detection is crucial for biomarker detection in research and diagnosis. Molecular tools for antibody detection and purification currently available consist of secondary antibodies as well as bacterial proteins such as Proteins A and G. Disadvantages of the current methods include the fact that secondary antibody production is expensive and may require the use of animals. In the case of Proteins A and G, they either do not recognize all IgG isotypes or require acidic elution conditions for antibody purification, which may lead to antibody denaturation. We have produced chimeric proteins, using an IgG binding protein recently discovered (TRIM21) linked to detection proteins such as streptavidin, and alkaline phosphatase. TRIM21 is a cytosolic protein that binds all IgG isotypes from many species. Bacterial expression, purification and refolding of TRIM21‐streptavidin as well as TRIM21‐alkaline phosphatase were successful. TRIM21‐streptavidin chimeric protein was incubated with biotinylated HRP or alkaline phosphatase for immunoassays. ELISA and western blotting experiments demonstrated that the chimeric proteins efficiently detect monoclonal and polyclonal antibodies from a wide range of species, including human, mouse, rat, dog, horse and bovine. Binding experiments are currently underway to determine the binding constants for TRIM21 chimeric proteins and IgG from different species. Future experiments will be performed using imobilized TRIM21 for antibody purification.Support or Funding InformationThis work was supported by the “ State University of Santa Catarina” (UDESC), “Fundação de Amparo a Pesquisa e Inovação do Estado de Santa Catarina” (FAPESC) and CNPQ.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.